FR2465782A1 - SELECTIVE ISOLATION MEDIUM OF CHOLERIC VIBRIONS, BASED ON MANNOSE - Google Patents

Abstract

THE OBJECT OF THE INVENTION IS A SELECTIVE ISOLATION MEDIUM FOR CHOLERA VIBRIONS. IT INCLUDES MANNOSE, A SULFONAPHTHALEIN COLOR, POLYMYXIN B, A SURFACTANT AND POTASSIUM TELLURITE; THIS MEDIUM HAS BOTH EXCELLENT DIFFERENTIABILITY AND SELECTIVITY AND IT IS ESPECIALLY EFFECTIVE TO DETECT CHOLERA BACTERIA FROM SUBSTANCE FROM THE ENVIRONMENT OR FROM CLINICAL MATERIALS FROM PATIENTS UNDER-HEALING, THAT IS CONTAINING A SMALL NUMBER OF CHOLERA BACTERIA. USE OF THIS MEDIUM FOR THE POSSIBLE DETECTION OF AN EARLY CHOLERA EPIDEMIC.

Description

The present invention relates to a new selective isolation medium.

tif for cholera vibrios.

  Vibrio or cholera bacteria belong to the vibrio genus and when they are infected with cholera, the patient suffers from disorders accompanied by diarrhea, nausea and exsiccosis. Due to the epidemic nature of this disease, it is frequently necessary to detect cholera vibrios in patients or in environments such as water, food, etc. Until now, the detection of vibrios or bacteria has been carried out

  in various selective isolation media such as thiosulfa-

  te-citrate de bile (abbreviated TCBS) and which is an agar-agar medium,

  in agar-agar medium for vibrios and the like.

  However, since a large number of non-agglutinable vibrios (NAG vibrios) belonging to the vibrio genus are known which are

  similar to each other with regard to the physiological properties

  the isolation medium must be able to allow the selective growth of only cholera vibrios (so-called medium selectivity) and it must also allow the formation of a colony characteristic of cholera vibrios (which the we

calls differentiability).

  The known media referred to above are formed to isolate and differentiate vibrion-like bacteria from clinical substances such as feces of patients with the use of sucrose and a pH indicator for give this environment the characteristic of differentiability and

  uses bile salts as selectivity conferring components.

  However, the detection of cholera vibrios by such media has the following disadvantages

  1) NAG vibrios other than cholera vibrios are allowed

  to develop and selectivity is therefore poor; 2) Many NAG vibrios are able to break down sucrose

  in the same way as the cholera vibrio and, under these conditions, the difference

  rentibility becomes mediocre; 3) the media serve to inhibit more or less the growth of cholera bacteria and thus face a difficulty in

  detection of cholera bacteria from materials originating from

  virulations or material from the patients themselves who are

  pathway of healing, that is, materials containing only a small amount of

  of cholera bacteria, or a difficulty of detection

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  cholera bacteria whose ability to grow has been weakened; and 4) the growing colony becomes resistant as a result of the presence of bile salts which may lead to erroneous results in the subsequent serum agglutination test. Thus it is found that the inspection of cholera bacteria using known media is complex and incorrect; in addition, one sometimes fails to be aware of the contamination by cholera vibrios,

  especially during an environmental inspection.

  Consequently, the main object of the invention is an isolation medium.

  selective for cholera vibrios characterized by both

  excellent differentiability and excellent selectivity.

  The subject of the invention is also a selective isolation medium for cholera vibrios, which medium is effective for detecting bacteria.

  cholera from substances from environments or subsi-

  clinical cases from patients in the process of re-establishment.

  is lying; that is, containing a small number of cholera bacteria.

  The stated objectives are achieved by mixing mannose and a

  sulfonaphthalein dye, as components conferring the difference between

  rability, as well as polymyxin B, a surfactant and

  potassium tellurite, as components conferring selectivity

you.

  More specifically, the invention envisages producing an isolation medium

  selectively for cholera vibrios, which comprises mannose, a sulfonaphthalein dye, polymyxin B, a surfactant

and potassium tellurite.

  Among the surfactants which must constitute one of the ingredients of the medium according to the invention, it is appropriate to mention ampholytic surfactants such as, for example, sodium laurelsulfate, sodium heptadecyl sulphate and the like. This agent is incorporated in the medium mainly to positively inhibit the growth of gram-positive bacteria. Polymyxin B is used to inhibit the growth of vibrion bacteria other than cholera bacteria and gram-negative bacteria. On the other hand, potassium tellurite is used to inhibit the growth of bacteria that are resistant to

  growth inhibitors mentioned above.

  Preferably, the concentrations of all these ingredients are determined as follows: Polymyxin B is present at 100,000 to 200,000 U / liter; the surfactant is present at 0.009 to 0.25% w / v; and potassium tellurite is present at 0.00008 at

  0.00015% w / v. The medium which contains polymyxin B, the surfactant

  potassium tellurite in the indicated concentrations of

  It does not exert any harmful effect on the growth of cholera bacteria but it prevents the growth of virtually all other bacteria, which means that its selectivity is first-rate. Only a few types of microorganisms can grow in the medium containing the ingredients listed, and of these, virtually no bacteria are found except cholera bacteria, which can break down mannose into an acid. As a result, it is easy to notice the presence of cholera bacteria by changing

  the color of the medium when incorporating therein the sulphate dye.

  fonaphthalein, whose color changes with a change in pH. Preferably the concentrations of these ingredients in the medium are determined so that the mannose represents more than 1.5% and

  preferably from 1.5 to 2.5% w / v and the sulfonaphthalein dye

0.002 to 0.008% w / v.

  Among the sulfonaphthalein dyes usable in the medium according to the invention, mention may be made of promothymol blue, thymol blue,

  cresol red, phenol red and the like.

  The medium according to the invention also contains, in addition to the ingredients which have been studied above, nutritional sources of the type usually used, such as peptone, meat extracts,

  sodium chloride etc., as well as agar-agar powder.

  To prepare the medium according to the invention, the indicated ingredients are dissolved in water with heating, the solution thus prepared is poured into a vessel and cooled to a solid state. In a preferred embodiment, all ingredients other than potassium tellurite are mixed and dried to obtain a dried powder, while potassium tellurite is placed in another container. In use, the dry powder is dissolved in purified water

  with heating and then a dilute aqueous solution of

  potassium rite (preferably in a concentration of about 0.1%); then we pour everything into a suitable container and cool

until solidification.

  To detect cholera bacteria using the medium of the invention, a sample to be tested is applied to the surface of the medium, cultivated for sixteen hours at 37 ° C. and observed visually; if the cholera bacteria are present, they appear in the form of

yellow colonies.

  The following examples serve to illustrate the invention without any

limit its scope.

EXAMPLE 1

  Table I: Composition of the Selective Isolation Medium According to the Invention Meat Extract Powder: 5.0 g Polypeptone: 10.0 g Sodium Chloride: 10.0 g D-Mannose: 20.0 g Agar Powder -agar: 15.0 g Polymyxin B: 180 000 U Sodium heptadecylsufate: 0.1 g Cresol red: 0.04 g Bromothymol blue: 0.04 g Potassium tellurite: 1.0 mg Purified water: 1000 All these ingredients are dissolved in the purified water with heating and each 20 ml portion of the solution thus prepared is placed in

  a Petri dish, cool and solify to prepare a medium.

EXAMPLE 2

  As well in the medium prepared in Example 1, that has a medium

  TCBS (thiosulfate-citrate bile) agar agar agar-agar agar-agar

  In addition, sewage containing cholera bacteria (approximately up to 1,000 cholera bacteria per milliliter of sewage) is added, cultured at 37 ° C for 16 hours and then visually observed. . Compositions of TCBS Medium and Agar Agar Medium

  vibrionary are shown in Tables II and III respectively.

  The results are given in Table IV.

  Table II: Composition of the TCBS agar medium per liter of

aqueous medium.

  Yeast Extract: 5.0 g Peptone: 10.0 g Sodium Citrate: 10.0 g Sodium Thiosulfate: 10.0 g Beef Bile: Sodium Cholate: Saccharose: Sodium Chloride: Ferric Citrate: Bromothymol Blue : Thymol blue: Agar-agar: pH: about 8.6 Table III: Composition of medium

of aqueous medium.

  Yeast Extract: 5.0 g Peptone: 5.0 g Sucrose: 12.5 g Sodium Taurocholate: 5.0 g Sodium Lauryl Sodium: 0.2 g Sodium Citrate: 8.0 g Sodium Thiosulfate: 8, 5 g Disodium phosphate: 7.5 g Ferric citrate: 3.0 g Sodium chloride: 10.0 g Cresol red: 0.02 g Water blue: 0.2 g Agar-agar: 15.0 g Vibrion agar per liter pH: 8.5 + 0.1 Table IV: Test results, 0 g 3.0 g, 0 g, 0 g 1.0 g 0.04 g 0.04 g, 0 g

  Sample Number of bacteria Medium Agar medium - Agar medium

  Cholera per ml according to agar TCBS agar vibrion-

  of water of invention the invention area 1 Several + (-) (+) - (++) 2 slightly more than 10 ++ (-) - (+) + (++)

3 100 +++ (-) - (+) + (++)

4 I 000 +++ (-) + (+) ++ (++)

  Notes: -: not observed +: I to 10 colonies observed ++: 11 to 99 colonies observed +++: 100 colonies or more observed The sign in parenthesis () indicates colonies of bacteria

  other than cholera bacteria.

EXAMPLE 3

  1 ml of the sample n 2 of Example 2 is inoculated with 10 ml of Monsur peptonic water (enrichment medium for cholera bacteria), the composition of which is indicated in Table V, and then

2 3

  cultivated for 8 hours at 37 C. This medium is diluted to 102, 10, 104, 105 and 106 times by volume. A predetermined quantity of each dilution is inoculated into the medium according to the invention, the TCBS medium and the agar-agar vibrioary medium, the respective compositions of which have been given in Tables I, II and III and the bacteria are cultured at 37 ° C. for sixteen hours to detect their growth state in the same way as in Example 2. The results are given in

Table VI.

  Table V: Monster peptone water composition Peptone: 10.0 g Sodium chloride: 10.0 g Sodium taurocholate: 5.0 g Sodium carbonate: 1.0 g Purified water: 1000 ml pH: 9, 0 - 9.2

Table VI

  Dilution Medium according to agar medium- Agar medium

  the invention TCBS agar vibrionary agar

102 +++ (-) ++ (-) ++ (+)

103 +++ (-) + (-) + (+)

104 ++ (-) - (-) + (+)

10 + (-) - (-) - (+)

I106- (-) - (-) - (+)

  The signs +++ ... (+) have the same meaning as in Example 2.

Claims (5)

  1. Selective isolation medium for cholera vibrios,   characterized in that it comprises mannose, a sulfonaphthalein dye,   polymyxin B, a surfactant and potassium tellurite.   2. Medium according to claim 1, characterized in that it comprises, relative to the total amount of the medium: 1.5 to 2.5% w / v of mannose, 0.002 to 0.008% w / v of dye of sulfonaphthalein, 000 to 200,000 U / liter polymyxin B, 0.009 to 0.25% w / v surfactant, and 0, 00008 to 0.00015% w / v tellurite tellurite potassium.   3. Medium according to claim 1 or 2, characterized in that the sulfonaphthalein dye is bromothynol blue, thymol blue,   cresol red or phenol red.   4. Medium according to claim 1 or 2, characterized in that   the surfactant is an ampholytic surfactant.   5. Medium according to claim 1 or 2, characterized in that the surfactant is sodium lauryl sulphate or heptadecyl sulphate. sodium.